Abstract: Manganese (Mn) is an essential element, but overexposure is cytotoxic and has adverse effects on neurological health. In humans, Mn-induced neurotoxicity generally occurs due to chronic exposure under occupational or environmental settings and resembles idiopathic Parkinson?s disease. In some cases, patients with compromised liver function due to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn- induced parkinsonism in the absence of high exposure. While the nutritional and clinical significance of Mn is established, cellular mechanisms of Mn homeostasis are still unknown. A breakthrough in our understanding of Mn metabolism came from the identification of a familial form of parkinsonism reported to occur due to mutations in SLC30A10. Findings in our lab have determined that SLC30A10 acts as the primary Mn efflux transporter protein to protect cells against Mn-induced toxicity. Interestingly, SLC30A10 disease-causing mutants from parkinsonian patients discussed above were unable protect against high Mn. As Mn is ubiquitous in the environment, our long term goal is to elucidate the role that SLC30A10 plays in metal-induced neurodegenerative processes, which in turn lead to Parkinson-like symptoms in patients. This gap in knowledge hinders treatment development and will persist if molecular mechanisms utilized by SLC30A10 are not understood. Our hypothesis is that SLC30A10 binds and transports Mn with higher affinity than other essential metals and that this activity is sensitive to cellular environment. We have recently identified residues of SLC30A10 that are required for Mn efflux activity. However, our studies used cell-based functional experiments and do not provide the molecular detail of their mechanistic involvement in Mn efflux activity. To shed light on this, experiments proposed here will determine the Mn transport mechanism of SLC30A10 using a combination of in vitro studies and physiologically relevant cell-based assays. In Aim 1 we will perform biochemical studies on purified SLC30A10 protein to reveal the mechanism of binding and transport of SLC30A10. First, isothermal titration calorimetry (ITC) will be used to determine the Mn binding coefficient (KD) of SLC30A10. Then a proteoliposome transport assay, with artificial membranes containing SLC30A10 will determine the Mn transport kinetics (KM and Vmax). We will perform a comparison of SLC30A10WT to SLC30A10 efflux mutants identified in our primary screens to elucidate residues directly involved in Mn binding. Aim 2 will then be performed in cell-based systems. Confocal microscopy will be employed to assess SLC30A10 function in primary neurons and a hepatic cell line. Quantitative metal measurement ICP-MS will be used to measure intracellular Mn content and corroborate microscopy findings. Taken together, the findings from this training plan will improve our understanding of cellular Mn homeostasis as it relates to neurotoxicity and provide biochemical data on SLC30A10 important for developing therapies against Mn toxicity.